Peptides having specificity for the lungs

ABSTRACT

The invention relates to a peptide, polypeptide, or protein that binds specifically to cells of the lung endothelium. The peptide, polypeptide, or protein can be a component of a viral capsid and can be used to lead a recombinant viral vector selectively to the lung endothelial tissue after systemic administration to a subject and to ensure tissue-specific expression of one or more transgenes there. The invention thus further relates to a recombinant viral vector, preferably an AAV vector, which comprises a capsid comprising the peptide, polypeptide, or protein according to the invention and which comprises at least one transgene packaged in the capsid. The viral vector is suitable in particular for the therapeutic treatment of a lung disorder or a lung disease. The invention further relates to cells and pharmaceutical compositions which comprise the viral vector according to the invention.

RELATED APPLICATIONS

This application is the National Stage of International PatentApplication No. PCT/EP2014/066892, filed Aug. 6, 2014, which is herebyincorporated by reference in its entirety, and which claims priority toGerman Patent Application No. DE 10 2013 215 817.3, filed Aug. 9, 2013.

SEQUENCE LISTING

The sequences listed in the accompanying Sequence Listing are presentedin accordance with 37 C.F.R. 1.822. The Sequence Listing is submitted asan ASCII computer readable text file, which is incorporated by referenceherein.

The invention relates to a peptide, polypeptide, or protein that bindsspecifically to cells of the lung endothelium. The peptide, polypeptide,or protein can be a component of a viral capsid and can be used to leada recombinant viral vector selectively to the lung endothelial tissueafter systemic administration to a subject and to ensure tissue-specificexpression of one or more transgenes there. The invention thus furtherrelates to a recombinant viral vector, preferably an AAV vector, whichcomprises a capsid comprising the peptide, polypeptide, or proteinaccording to the invention and which comprises at least one transgenepackaged in the capsid. The viral vector is suitable in particular forthe therapeutic treatment of a lung disorder or a lung disease. Theinvention further relates to cells and pharmaceutical compositions whichcomprise the viral vector according to the invention.

BACKGROUND OF THE INVENTION

Pulmonary hypertension is a serious chronic lung disease which regularlyleads to death if untreated. The term applies to diseases of variouscauses, which are characterized by a structural change in the pulmonaryvasculature, and in which there is an increase in blood pressure in thepulmonary arterial system to more than 25 mm Hg [1]. This usuallyresults, in affected patients, in stress-dependent shortness of breath,and general loss of capacity. Disease progression leads to a narrowingof vessels resulting from a transformation (remodeling) and thickeningof all three layers of the vessel wall, i.e. intima, media andadventitia [2]. This often leads to resting dyspnea, global respiratoryinsufficiency, and the congestive syndromes associated with right-sidedheart failure and in the long-term to heart failure. Pulmonary arterialhypertension is a particularly severe form of pulmonary hypertension inwhich the median survival from diagnosis is only about three years [3],and diagnosis is often made very late due to the initially mildsymptoms.

Several animal models that functionalize different diseasecharacteristics are available for investigating the mechanisms ofpulmonary hypertension, and for pre-clinical treatment studies. Theseinclude both inducible models (hypoxia, monocrotaline, or antigens, forexample) and transgenic models, wherein the selection of a suitablemodel depends on the research question being examined [4].

Not all possible causes of the various forms of pulmonary arterialhypertension have been explained to date. Nevertheless, there areseveral well-known and therapeutically relevant factors. For example, incases of idiopathic pulmonary arterial hypertension, the increasedrelease of vasoconstrictive factors is discussed [5-7], while in manycases of familial pulmonary arterial hypertension, mutations of BMPR2[8] or the Activin receptor-like kinase 1 (ALK1) gene [9] are consideredlikely causes.

The development of new therapeutic options for the treatment ofpulmonary hypertension or pulmonary arterial hypertension is an urgentneed. Such a development could be the transfer of therapeutic genes intolung tissue, and more particularly into the pulmonary endothelium.Vectors that allow a specific and efficient gene transfer into thepulmonary endothelium have not yet been described in the prior art. Genetherapy using viral vectors is a promising treatment option for diseasesthat do not respond at all, or not adequately, to conventionaltreatment. This approach is based on the introduction of therapeuticgenes into the organism being treated, by means of viruses which havebeen modified in such a manner that they have the sequence of thecorresponding gene in their genome. Viral vectors which have alreadybeen used in a gene therapy regimen for gene therapy approaches arebased on retroviruses, lentiviruses, adenoviruses and adeno-associatedviruses.

Adeno-associated viruses (AAVs) are promising candidates for use inclinical practice because they are classified as relatively safe. AAVvectors are able to introduce a transgene into a tissue and express thegene stably and efficiently in the tissue. At the same time, thesevectors have no known pathogenic mechanisms [10]. Of particularimportance for clinical use are the AAV vectors of serotype 2 (AAV2),which are considered to be particularly well investigated. After the AAVvectors are introduced, the transgenes can be incorporated in differentforms in the transfected cells—for example as episomal, single- ordouble-stranded DNA. Concatamer forms of the DNA have also beendemonstrated in transduced cells.

The genome of AAV2 is formed by a linear, single-stranded DNA moleculeof approximately 4700 nucleotides in length and has inverted terminalrepeats (ITRs) at both ends. The genome also includes two large openreading frames which are called the replication region (rep) and thecapsid region (cap). The replication region encodes proteins that arerequired as part of the virus replication. The capsid region, however,encodes for the structural proteins VP1, VP2 and VP3, which make up theicosahedral capsid of the virus.

Like most vectors which have gene therapy applications and are known inthe prior art, however, wild-type AAV vectors, such as the AAV2 vectorsdescribed above, do not possess sufficient specificity for a particulartissue, and infect a wide variety of cell types. As such, systemicadministration of wild-type vectors leads to insufficient transductionof lung tissue, and severe immune reactions are expected in thetreatment subject due to the unwanted transduction of other tissues.Progress in the development of viral vectors which have an increasedspecificity for particular organs has been made in the past by the useof peptide ligands, which are able to direct the vectors to a particularorgan [11-12]. It has been shown that certain peptide ligands bringabout a “homing” to various organs such as the brain.

Reading [13] describes a method which enables the screening fortropism-modified capsids of AAV2 in randomized peptide libraries. Fromthese libraries, vectors can be isolated which specifically transduce adesired cell type in vitro. However, it has been surprisingly found thatcapsids selected in this manner are often unsuitable for use in vivobecause they lack the necessary specificity in animal models [14].

There remains a great need for agents that are able to modulate thetropism of viral vectors and thus ensure adequate cell or tissuespecificity to enable targeted delivery of a viral vector into the lung.Such vectors enable specific expression of therapeutic genes in lungtissue, for the corresponding, effective treatment of diseases and/ordisorders of the lungs.

The present invention makes available viral vectors for targeted genetransfer to the lungs. The viral vectors according to the inventionexpress on their capsid surface a previously unknown amino acid sequencethat is specifically recognized in vivo by receptors on the endothelialtissue of the lung. As such, the viral vectors of the present inventionspecifically transduce the lung tissue of a patient following systemicadministration to the same.

The viral vectors according to the invention also enable a strong andpersistent expression of a transgene in the endothelial cells of thelung with only minor immune response, and are therefore particularlysuitable for gene therapy treatments of certain pulmonary disordersand/or lung diseases. After transfection, the AAV vectors only instigatea minor immune response in the host and are therefore particularlysuitable for gene therapy.

DESCRIPTION OF THE INVENTION

In the present invention, various lung-specific sequences wereidentified by selection in a randomized AAV2 heptamer peptide library.Beginning with the identified peptide sequence ESGHGYF (SEQ ID NO: 2),the recombinant viral vector rAAV2-ESGHGYF (SEQ ID NO:9) was prepared,in which the peptide sequence ESGHGYF (SEQ ID NO: 2) is expressed as apartial sequence of the capsid protein VP1. Subsequently, the vectorrAAV2-ESGHGYF (SEQ ID NO:9) was administered intravenously to mice, anda unique specificity of the vector for lung endothelial tissue wasobserved both in vitro and in vivo. The specificity was demonstrated bystaining with CD31, as described in the examples below. Moreover, byreplacing the amino acids glutamic acid (E) and serine (S) at theN-terminus of the peptide, it was possible to use an alanine-scan toshow that these two amino acids are not relevant for the specificity ofthe transduction. As such, only the core structure GHGYF (SEQ ID NO: 1)is responsible for the lung specificity.

The present invention therefore provides various lung-specific peptidesequences which are particularly suited for directing therapeutic agentssuch as viral vectors to the lungs of a subject being treated.

A first aspect of the present invention accordingly relates to apeptide, polypeptide, or protein that binds specifically to endothelialcells in the lungs, wherein the peptide, polypeptide, or proteincomprises the amino acid sequence of SEQ ID NO: 1. The peptide,polypeptide, or protein preferably comprises the amino acid sequence ofSEQ ID NO: 2 or a variant thereof which differs from the amino acidsequence of SEQ ID NO: 2 by the modification of at least one of the twoN-terminal amino acids. The peptide, polypeptide or protein according tothe invention preferably binds to endothelial cells of the lung—inparticular, the human lung.

In another aspect, the invention relates to a peptide, polypeptide orprotein which binds specifically to endothelial cells of the lung,wherein the peptide, polypeptide or protein comprises the amino acidsequence of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5,

In the context of the present disclosure, the term “peptide” refers to alinkage of 2-10 amino acids which are connected to each other by apeptide bond. The term “polypeptide” refers to a linkage of 11-100 aminoacids that are connected to each other by a peptide bond. Polypeptideswith more than 100 amino acids are referred to herein as a “protein.” Ina particularly preferred embodiment according to the invention, thelung-specific peptide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or thevariant of SEQ ID NO: 2, according to the invention is a part of acapsid protein of a virus. This means that the lung specific sequence ispresent as part of a capsid protein of the virus. To produce such capsidproteins, a corresponding nucleotide sequence which codes for thelung-specific peptide is cloned into the region of the virus genomewhich codes for a capsid protein of the virus. If the lung specificsequence is expressed as part of a capsid protein, it can be presentedin many copies across the surface of the viral vector.

The capsid protein is preferably one which is derived from anadeno-associated virus (AAV). The AAV can be any of the serotypesdescribed in the prior art, wherein the capsid protein is preferablyderived from an AAV of one of the serotypes 2, 4, 6, 8 and 9. A capsidprotein of an AAV of serotype 2 is particularly preferred.

The capsid of the AAV wild-type is made up of the capsid proteins VP1,VP2 and VP3, which are encoded by the overlapping cap region. All threeproteins have the same C-terminal region. The capsid of AAV comprisesabout 60 copies of the proteins VP1, VP2 and VP3, expressed in a ratioof 1:1:8. If a nucleotide sequence coding for one of the lung-specificpeptides described herein is cloned at the C-terminus into the readingframe of VP1 (i.e., in the region that is identical in all threeproteins), it can be expected that theoretically 60 of the specificpeptides can be found on the capsid surface.

If an AAV vector in the context of the present invention is modified,the nucleotide sequence coding for the lung-specific peptide is thencloned into the cap region at the 3′ end of the genome. The geneencoding the lung-specific peptide sequence can be cloned into thegenomic sequence of one of the capsid proteins VP1, VP2 or VP3. Thecapsid proteins of AAV2 are illustrated by way of example in SEQ ID NO:6 (VP1), SEQ ID NO: 7 (VP2), and SEQ ID NO: 8 (VP3).

In one particularly preferred embodiment according to the invention, thegene encoding the lung-specific peptide sequence is cloned into thereading frame of a VP1 gene, preferably in the VP1 gene of AAV2 shown inSEQ ID NO: 6. It should be noted in this case that the insertion of thecloned sequence does not lead to any change of the reading frame, nor toa premature termination to the translation. The methods required for theabove are readily apparent to a person skilled in the art.

In all three capsid proteins of AVV, sites have been identified at whichpeptide sequences can be inserted for the homing function [15-20]. Amongother things, the arginine which occurs in the VP1 of AAV2 at position588 (R588) has specifically been proposed for the insertion of a peptideligand [21-22]. This amino acid position of the viral capsid isapparently involved in the binding of AAV2 to its natural receptor. Ithas been suggested that R588 is one of four arginine residues whichmediates the binding of AAV2 to its natural receptor [23-24]. Amodification in this region of the capsid weakens the natural tropism ofAAV2, or eliminates it completely.

Accordingly, it is particularly preferred according to the inventionthat the inventive lung specific peptide sequence of SEQ ID NO: 1 or SEQID NO: 2 (or their variants), or of one of the lung-specific peptidesequences of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, is present ininserted form in the region of the amino acids 550-600 of the VP1protein of AAV2, in particular the protein of SEQ ID NO: 6. Even morepreferably, the lung-specific peptide sequence is present in insertedform in the region of amino acids 560-600, 570-600, 560-590, 570-590 ofthe VP1 protein.

Thus, the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or theirvariants (or alternatively, the peptide sequences of SEQ ID NO: 3, SEQID NO: 4 or SEQ ID NO: 5), adjoin, by way of example directly behind,one of the following amino acids of the VP1 protein, particularly theprotein of SEQ ID NO: 6: 550, 551, 552, 553, 554, 555, 556, 557, 558,559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572,573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586,587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599 or 600.It is particularly preferred that the amino acid sequence of SEQ ID NO:1 or SEQ ID NO: 2 or its variants (or alternatively, the peptidesequences of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5) follows theamino acid 588 of the VP1 protein of SEQ ID NO: 6, as shown in theexamples. It is possible in this case that one or more, and particularlyup to 5 (for example, 1, 2, 3, 4 or 5) amino acids which are the resultof the cloning are situated between the arginine residue at position 588and the first amino acid of SEQ ID NO: 1 or SEQ ID NO: 2, or the variantof SEQ ID NO: 2. Likewise, one or more, and particularly up to 5 (i.e.,1, 2, 3, 4, or 5) amino acids can be situated behind the last amino acidof SEQ ID NO: 1 or SEQ ID NO: 2, or the variant of SEQ ID NO: 2.

The sites and regions in the amino acid sequence of the capsid proteinindicated above for VP1 apply analogously to the capsid proteins VP2 andVP3 of AAV2. Because the three capsid proteins VP1, VP2 and VP3 of AAV2differ only by the length of the N-terminal sequence and accordinglyhave an identical C-terminus, a person skilled in the art will have noproblem making a sequence comparison to identify the sites indicatedabove, for the insertion of the peptide ligands, in the amino acidsequences of VP1 and VP2. As such, the amino acid 588 in VP1 correspondsto position R451 of VP2 (SEQ ID NO: 7) and/or position R386 of VP3 (SEQID NO: 8).

SEQ ID NO: 9 shows an example of the sequence of the VP1 protein of AAV2after introduction of the peptide sequence of SEQ ID NO: 2. Due to thecloning, the capsid protein has two additional amino acids which do notoccur in the native sequence of the VP1 protein of AAV2. As such, thepeptide sequence of SEQ ID NO: 2 is flanked at its N-terminus by aglycine in position 589, and at its C-terminus by an alanine in position597. In addition, the asparagine at position 587 of the native sequenceis replaced with a glutamine.

In one particular embodiment, the present invention therefore alsorelates to a capsid protein which comprises or consists of:

-   -   (a) the amino acid sequence of SEQ ID NO: 9;    -   (b) an amino acid sequence which is at least 80% identical to        the amino acid sequence of SEQ ID NO: 9; or    -   (c) a fragment of one of the amino acid sequences defined in (a)        or (b).

In a further aspect, the invention is directed to a viral capsid, whichcomprises a peptide, polypeptide or protein which specifically binds tocells of the lung, and which has the amino acid sequence of SEQ ID NO: 1or SEQ ID NO: 2 or a variant of SEQ ID NO: 2 as described above (oralternatively, the peptide sequences of SEQ ID NO: 3, SEQ ID NO: 4 orSEQ ID NO: 5).

Furthermore, the present invention also provides a nucleic acid encodinga peptide, polypeptide or protein as described above. A nucleic acidwhich encodes a capsid protein which comprises a peptide, polypeptide orprotein according to the invention as described above, is likewiseprovided. Preferably, the nucleic acid coding for the capsid proteincomprises the nucleotide sequence of SEQ ID NO: 10 or a nucleotidesequence derived from the same, with at least 80% sequence identity. Aplasmid comprising such a nucleic acid is also provided.

In yet another aspect, the invention relates to a recombinant—i.e.,produced by means of genetic engineering techniques—viral vector, havinga capsid and at least one transgene packaged therein, wherein the capsidcomprises at least one capsid protein having the amino acid sequence ofSEQ ID NO: 1 or SEQ ID NO: 2, or of a variant of SEQ ID NO: 2 whichdiffers from the amino acid sequence of SEQ ID NO: 2 by modification ofat least one of the two N-terminal amino acids. Alternatively, thecapsid protein can also comprise the amino acid sequence of SEQ ID NO:3, SEQ ID NO: 4 or SEQ ID NO: 5. The recombinant viral vector can be arecombinant AAV vector—for example, of serotype 2, 4, 6, 8, 9. AAVvectors of serotype 2 are particularly preferred.

The different AAV serotypes differ mainly by their natural tropism. Assuch, wild-type AAV2 binds more readily to alveolar cells than to lungepithelial cells, while AAV5, AAV6 and AAV9 are capable of greatertransduction of epithelial cells of the respiratory tract. A personskilled in the art can take advantage of these natural differences inthe specificity of the cells to further amplify the specificity mediatedby the peptides according to the invention for certain cells or tissues.At the nucleic acid level, the various AAV serotypes are highlyhomologous. For example, serotypes AAV1, AAV2, AAV3 and AAV6 are 82%identical on the nucleic acid level [25]. The capsid of the viralvectors according to the invention comprises one or more transgenes. Agene which has been introduced by genetic engineering into the genome ofthe vector is termed a transgene. The transgene (or transgenes) can beDNA or RNA. Preferably, it is single-stranded DNA (ssDNA) ordouble-stranded DNA (dsDNA), such as genomic DNA or cDNA. Preferably,the transgene which is to be transported with the aid of the recombinantviral vector according to the invention is a human gene. Suitabletransgenes include, for example, therapeutic genes to replace adysfunctional gene in patients. They can also be genes which are notexpressed in the corresponding target tissue, or are only expressed toan insufficient extent. In one preferred embodiment, the transgene whichis expressed from the viral vector according to the invention is a geneencoding a nitric oxide synthase (NOS) or the bone morphogenic proteinreceptor 2 (BMPR2). In the case of NOS, endothelial NOS (eNOS) orinducible NOS (iNOS) can be encoded. The genes encoding eNOS and iNOScan be used for the treatment of a pulmonary disorder or pulmonarydisease in a patient—in particular pulmonary hypertension or pulmonaryarterial hypertension.

In a further embodiment, the transgene encodes a radioprotective proteinsuch as a radioprotective enzyme. A major limitation of radiotherapy inthe treatment of cancer is the radiation-induced damage to the normaltissue, which often makes a reduction in radiation dose, an interruptionof the treatment regimen, or even a complete abandonment of this form oftherapy necessary. The lung is a therapeutically particularly relevantsite of tumor growth but at the same time a particularly radiosensitiveorgan, which is why primary tumors there often can only be treated to alimited extent with radiotherapy, and diffuse tumor growth usuallycannot be treated at all with radiotherapy. With the help oflung-specific vectors of the present invention, the healthy tissuesurrounding the malignant tissue can be protected by targeted expressionof radioprotective proteins. In one preferred embodiment, the transgenewhich is introduced into the healthy lung tissue is a manganesesuperoxide dismutase (MnSOD) which catalyzes the conversion ofsuperoxide anions—one of the critical factors in radiation-inducedtoxicity—to hydrogen peroxide. A further embodiment proposed hereinvolves the kinase domains of the ataxia telangiectasia mutant (ATM)gene, which contributes to the repair of DNA damage caused by radiation.In a further preferred embodiment, both genes are introduced by means ofthe presently described vectors into the patient undergoing treatment.

In a further embodiment, the transgene encodes for humanalpha-1-antitrypsin. The lack of sufficient quantities ofalpha-1-antitrypsin is the cause of Laurell-Eriksson syndrome, ahereditary metabolic disease which can lead to emphysema or cirrhosis.Alpha-1-antitrypsin is required for the regulation of the activity ofproteases in the serum. The lack of this inhibitor leads to increasedproteolysis in the serum and accordingly to the severe sequelae namedabove.

The viral vectors of the present invention are particularly suitable foruse in a method for therapeutic treatment of diseases of the lung. Lungdisorders in the context of the invention include, in particular, allkinds of vascular diseases of the lung such as pulmonary hypertension,as well as lung tumors, alpha-1-antitrypsin deficiency (A1AD), andothers. For example, lung tumors which are suitable for treatment usingthe vectors according to the invention include small cell lung cancer(SCLC), squamous-cell carcinoma, adenocarcinoma, and large cell lungcarcinoma. In one preferred embodiment, the viral vectors of the presentinvention are used for therapeutic treatment of pulmonary hypertensionor pulmonary arterial hypertension.

In yet another embodiment, the transgene encodes an antitumor agent,such as a tumor suppressor protein, or an immunomodulator such as acytokine (such as interleukin 1 to 4, gamma-interferon, p53), which isintended to be transported selectively to the lung tissue of thepatient.

The vectors can also be used to transport antisense-RNA, ribozymes, orthe like into the endothelial tissue of the lung. Furthermore, vectorsaccording to the invention can also comprise transgenes encodingsecretory proteins that are intended for systemic administration in thebloodstream. Such secretory proteins can be efficiently deposited in thebloodstream via the pulmonary capillary bed, which is part of thecardiovascular system.

As used herein, the term “subject” indicates any human or animalorganism that can be infected by AAV vectors. Preferably, the subjectbeing treated is a mammal such as a human, a primate, a mouse or a rat.In one preferred embodiment, the subject to be treated is a human. Aftertransfection into the subject, the vector brings about a site-specificexpression of the transgene in the cells of the lung endothelium.

The transgene can be present in the viral vector in the form of anexpression cassette, which in addition to the sequence of the transgeneto be expressed comprises further elements necessary for expression,such as a suitable promoter which controls the expression of thetransgene after infection of the appropriate cells. Suitable promotersinclude, in addition to the AAV promoters such as the cytomegalovirus(CMV) promoter or the chicken beta actin/cytomegalovirus hybrid promoter(CAG), an endothelial cell-specific promoter such as the VE-cadherinpromoter, as well as steroid promoters and metallothionein promoters. Inone particularly preferred embodiment, the transgene according to theinvention comprises a pulmonary endothelium-specific promoter which isconnected by a functional bond to the transgene to be expressed. In thisway, the specificity of the vectors according to the invention can befurther increased for lung endothelium cells. As used herein, apulmonary endothelium-specific promoter is a promoter whose activity inlung endothelial cells is at least 2-fold, 5-fold, 10-fold, 20-fold,50-fold or 100-fold higher than in a cell which is not a pulmonaryendothelium cell. Preferably, this promoter is a human promoter. Theexpression cassette can also include an enhancer element for increasingthe expression levels of exogenous protein to be expressed. Furthermore,the expression cassette can include polyadenylation sequences, such asthe SV40 polyadenylation sequences or polyadenylation sequences ofbovine growth hormone.

The viral vectors according to the invention can, preferably as part ofone of their capsid proteins, comprise the amino acid sequence of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.Alternatively, variants of the amino acid sequence of SEQ ID NO: 2 canbe used, the same differing from the amino acid sequence of SEQ ID NO: 2by the modification of at least one of the two N-terminal amino acids.The modification can be a substitution, deletion or insertion of aminoacids, as long as the variant retains the ability to communicate, aspart of the capsid, the specific binding of the vector to the receptorstructures of endothelial cells of the lung. The invention thereforealso extends to variants of the sequence of SEQ ID NO: 2 in which one ofthe two N-terminal amino acids of SEQ ID NO: 2 has been changed. Thesevariants, which are within the scope of the invention, therefore have asequence identity of more than 85% to the amino acid sequence shown inSEQ ID NO: 2 when the sequences are compared using the programs GAP orBESTFIT. These computer programs for determining amino acid sequenceidentity are sufficiently known in the art.

The variant of the sequence of SEQ ID NO: 2 can be based on thesubstitution of one or two of the N-terminal amino acids—that is, one orboth N-terminal amino acids can be replaced with another amino acid.Preferably, the substitution by which the variants of the amino acidsequence in SEQ ID NO: 2 differ is a conservative substitution—i.e., asubstitution of one amino acid by an amino acid of similar polaritywhich gives the peptide similar functional properties. Preferably, thesubstituted amino acid is from the same group of amino acids as theamino acids which are replaced. For example, a hydrophobic residue canbe replaced with another hydrophobic residue, or a polar residue byanother polar residue. Functionally similar amino acids which can beexchanged for each other by a conservative substitution include, forexample, non-polar amino acids such as glycine, valine, alanine,isoleucine, leucine, methionine, proline, phenylalanine, and tryptophan.Examples of uncharged polar amino acids are serine, threonine,glutamine, asparagine, tyrosine, and cysteine. Examples of charged,polar (acidic) amino acids include histidine, arginine and lysine.Examples of charged, polar (basic) amino acids include aspartic acid andglutamic acid.

Those amino acid sequences in which an amino acid has been inserted intothe region of the two N-terminal amino acids of SEQ ID NO: 2 are alsoconsidered variants of the amino acid sequence shown in SEQ ID NO: 2.Such insertions can in principle be carried out as long as the resultingvariant retains its ability to bind specifically to endothelial cells ofthe lung. In addition, in the present context, those proteins in whichone of the two N-terminal amino acids of SEQ ID NO: 2 is missing areconsidered to be variants of the amino acid sequence shown in SEQ ID NO:2. This requires in turn that the correspondingly deleted variant bindsspecifically to endothelial cells of the lung.

Also encompassed by the invention are lung endothelium-specific variantsof the amino acid sequence shown SEQ ID NO: 2, which are structurallymodified at one or both N-terminal amino acids—by way of example byintroducing a modified amino acid. According to the invention, thesemodified amino acids can be amino acids that have been modified bybiotinylation, phosphorylation, glycosylation, acetylation, branchingand/or cyclization.

Viral vectors with capsids which comprise one of the peptide sequencesaccording to the invention or comprise a variant thereof as definedabove bind specifically to endothelial cells of the lung. As usedherein, a “specific” binding of the vectors according to the inventionmeans that the vectors accumulate after systemic administration mainlyon or in the endothelial cells of the lung. This means that more than50% of the originally administered vector genomes accumulate in theendothelial cells, or in the area of the endothelial cells, of thelungs, while less than 50% accumulate in other cells or tissues (such asin the spleen or liver), or the area thereof. It is preferred that morethan 60%, 70%, 80%, 90%, 95%, or even more than 99% of the originallyadministered vector genomes accumulate in, or in the region of, theendothelial cells of the lung. Specific binding of the vectors can alsobe determined via the expression of the transgene. In the case of viralvectors which bind specifically to endothelial cells of the lung, morethan 50% of the total expression of the transgene occurs in, or in theregion of, the endothelial cells of the lung, while less than 50% of theexpression can be observed in, or in the region of, other tissues. It ispreferred, however, that more than 60%, 70%, 80%, 90%, 95%, or even morethan 99% of the total measured expression of the transgene occurs in, orin the region of, the endothelial cells of the lungs.

A person skilled in the art will easily be able to determine thespecific binding of the vectors according to the invention toendothelial cells of the lung and the expression of the transgeneintroduced using the vectors. Methods for measuring the specificity oftransduction and expression, suitable for this purpose, are shown in theexamples below.

It is also preferable according to the invention that vectors whosecapsids comprise variants of the amino acid sequence shown in SEQ ID NO:2 have at least about 50% of the binding activity of a correspondingviral vector with a capsid which has the amino acid sequence shown inSEQ ID NO: 2. It is even more preferred that the variants have about60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the binding activityof a corresponding viral vector whose capsid has the amino acid sequenceshown in SEQ ID NO: 2. The binding activity of the vectors can bemeasured with the aid of in vitro assays, as described in the includedexamples.

Preferably, the binding activity of the vectors according to theinvention, as can be determined by distribution of the vector genomes orexpression of the transgene, for endothelial cells of the lung is atleast 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 75-fold, 100-fold,150-fold, 200-fold, 250-fold, 500-fold, 600-fold, 700-fold, 800-fold,900-fold, 1000-fold, 2000 fold, 5000-fold, or 10,000-fold higher thanthe binding activity for a control cell which is not a lung endotheliumcell (such as a spleen or liver cell).

The invention also relates to a cell that comprises a peptide,polypeptide or protein according to the invention, a nucleic acidencoding the same, a plasmid comprising such a nucleic acid, or arecombinant AAV vector as described above. It is preferably a human cellor cell line.

In one embodiment, a cell has been, for example, obtained from a humansubject by biopsy and then transfected with the viral vector in an exvivo procedure. The cell can then be re-implanted in the subject, or besupplied in other ways to the subject—for example by transplantation orinfusion. The likelihood of rejection of transplanted cells is lowerwhen the subject from which the cell was derived is genetically similarto the subject to which the cell is administered. Preferably, therefore,the subject to whom the transfected cells are supplied is the samesubject from which the cells were previously obtained. The cell ispreferably a human lung cell, particularly a cell of the human pulmonaryendothelium. The cell to be transfected can also be a stem cell, such asa human adult stem cell. It is particularly preferred according to theinvention that the cells to be transfected are autologous cells thathave been transfected ex vivo with the viral vector according to theinvention, for example the recombinant AAV2 vector described above. Thecells are preferably used in a method for treating a pulmonary disorderor a lung disease in a subject.

In another aspect, the invention relates to a method for producing anAAV vector in which a plasmid is used which encodes a capsid protein,the same comprising the amino acid sequence of SEQ ID NO: 1 or SEQ IDNO: 2, or a variant thereof. Alternatively, the vector can also comprisethe amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.The basic method of producing recombinant AAV vectors comprising atransgene to be expressed is described in the prior art sufficiently[28]. HEK 293-T cells are transfected with three plasmids. A firstplasmid comprises the cap and rep regions of the AAV genome, but thenaturally occurring inverted repeats (ITRs) are missing. The cap regionof this plasmid comprises a gene encoding at least one modified capsidprotein, i.e., a gene which comprises the peptide sequence according tothe invention. A second plasmid comprises a transgene expressioncassette which is flanked by the corresponding ITRs, which constitutethe packaging signal. The expression cassette is therefore packaged intothe capsid in the course of the assembly of the viral particles. Thethird plasmid is an adenoviral helper plasmid, on which are encoded thehelper proteins E1A, E1B, E2A, E4orf6, VA, which are required for AAVreplication in the HEK 293-T cells.

Conditions which allow the accumulation and purification of therecombinant vectors according to the invention are known in the art. Thevectors according to the invention can be purified, for example, by gelfiltration processes—for example using a Sepharose—desalinated, andsubsequently purified by filtration. Other purification methods canfurther comprise a cesium chloride or iodixanol gradientultracentrifugation process. Purification reduces potentiallydetrimental effects in the subject to which the adeno-associated viralvectors are administered. The administered virus is substantially freeof wild-type and replication-competent virus. The purity of the viruscan be checked by suitable methods such as PCR amplification.

In a further aspect, the invention provides a pharmaceutical compositioncomprising a viral vector of the present invention, particularly an AAVvector. The viral vector in this case is administered in atherapeutically effective amount to the patient, i.e., in an amountsufficient to considerably improve at least one symptom of the lungdysfunction or lung disease being treated, or to prevent the progressionof the disease. Symptoms that are regularly associated with a lungdisease include cough, fever, chest pain, hoarseness and difficultybreathing. A therapeutically effective amount of the vector according tothe invention causes a positive change in one of the mentioned symptoms,i.e., a change which results in the phenotype of the affected subjectapproximating the phenotype of a healthy subject who does not sufferfrom a pulmonary disease.

In one preferred embodiment according to the invention, theadministration of the viral vector occurs in an amount which leads to acomplete or substantially complete healing of the lung dysfunction orlung disease. The pharmaceutical composition accordingly comprises atherapeutically effective dose of the vector according to the invention.A therapeutically effective dose will generally be non-toxic for thesubject who undergoes the treatment.

The exact amount of viral vector which must be administered to achieve atherapeutic effect depends on several parameters. Factors that arerelevant to the amount of viral vector to be administered are, forexample, the route of administration of the viral vector, the nature andseverity of the lung disease, the disease history of the patient beingtreated, and the age, weight, height, and health of the patient to betreated. Furthermore, the expression level of the transgene which isrequired to achieve a therapeutic effect, the immune response of thepatient, as well as the stability of the gene product are relevant forthe amount to be administered. A therapeutically effective amount of theviral vector can be determined by a person skilled in the art on thebasis of general knowledge and the present disclosure.

The viral vector is preferably administered in an amount correspondingto a dose of virus in the range of 1.0×10¹⁰ to 1.0×10¹⁴ vg/kg (virusgenomes per kg body weight), although a range of 1.0×10¹¹ to 1.0×10¹³vg/kg is more preferred, and a range of 5.0×10¹¹ to 5.0×10¹² vg/kg isstill more preferred, and a range of 1.0×10¹² to 5.0×10¹² is still morepreferred. A virus dose of approximately 2.5×10¹² vg/kg is mostpreferred.

The amount of the viral vector to be administered, such as the AAV2vector according to the invention, for example, can be adjustedaccording to the strength of the expression of one or more transgenes.

The viral vector of the present invention, such as the preferred AAV2vector according to the invention, for example, can be formulated forvarious routes of administration—for example, for oral administration asa capsule, a liquid or the like. However, it is preferred that the viralvector is administered parenterally, preferably by intravenous injectionor intravenous infusion. The administration can be, for example, byintravenous infusion, for example within 60 minutes, within 30 minutesor within 15 minutes. It is further preferred that the viral vector isadministered locally by injection to the lung during a surgery.Compositions which are suitable for administration by injection and/orinfusion typically include solutions and dispersions, and powders fromwhich corresponding solutions and dispersions can be prepared. Suchcompositions will comprise the viral vector and at least one suitablepharmaceutically acceptable carrier. Suitable pharmaceuticallyacceptable carriers for intravenous administration includebacteriostatic water, Ringer's solution, physiological saline, phosphatebuffered saline (PBS) and Cremophor EL™. Sterile compositions for theinjection and/or infusion can be prepared by introducing the viralvector in the required amount into an appropriate carrier, and thensterilizing by filtration. Compositions for administration by injectionor infusion should remain stable under storage conditions after theirpreparation over an extended period of time. The compositions cancontain a preservative for this purpose. Suitable preservatives includechlorobutanol, phenol, ascorbic acid and thimerosal. The preparation ofcorresponding formulations and suitable adjuvants is described, forexample, in “Remington: The Science and Practice of Pharmacy,”Lippincott Williams & Wilkins; 21st edition (2005).

In a further aspect, the invention relates to a method for thetherapeutic treatment of a pulmonary disorder or a pulmonary disease,wherein a viral vector according to the invention, preferably an AAVvector as described above, is administered to a subject. The vectorcomprises a capsid which has at least one capsid protein having theamino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant asdescribed above of SEQ ID NO: 2. Alternatively, the vector can alsocomprise the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ IDNO: 5. The viral vector further comprises a transgene, for example atherapeutic gene, which is useful for the treatment of the pulmonarydisorder or the lung disease. After administration to the subject beingtreated, preferably by systemic administration such as intravenousinjection or infusion, for example, the vector brings about the specificexpression of the gene in the endothelial cells of the lung.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the in vivo selection method of the AAV-peptide libraryused according to the invention.

FIG. 2 shows the sequences of the lung-specific peptides identified bymeans of the selection method. After the fourth round of selection, atotal of four different sequences were identified.

FIG. 3 shows the measurement of the expression of luciferase 14 daysafter systemic administration of recombinant AAV vectors in mouse organlysates. A: wild-type AAV2 vector (upper panel) and the insertioncontrol AAV2-CVGSPCG (SEQ ID NO:21, middle panel) induce mainlyheart-specific expression. AAV2-ESGHYGF (SEQ ID NO:9, lower panel)induces a strong expression of luciferase, which is simultaneouslylung-specific. B: Comparison of the expression levels of wild-type AAV2,AAV2-CVGSPCG (SEQ ID NO:21) and AAV2-ESGHYGF (SEQ ID NO:9) vectors inthe heart (upper panel), liver (middle panel) and lung (lower panel).AAV2-ESGHYGF (SEQ ID NO:9) has a greatly attenuated induction ofexpression in the heart and liver and a significant increase inexpression of luciferase in the lung. Mean values are shown with theirstandard deviation. One-Way ANOVA. p<0.05=*; p<0.01=**; p<0.001=*** forn=3.

FIG. 4 shows a long-term expression analysis in mouse after systemicadministration of recombinant AAV2-ESGHYGF (SEQ ID NO:9) vector.Repeated measurements using the IVIS® 200 Imaging System exhibit stablegene expression in the lung over a period of 168 days (n=1).

FIG. 5 shows the distribution of recombinant AAV vectors after systemicadministration of 5×10¹⁰ gp/mouse by quantitative real-time PCR. A:Distribution of AAV2-ESGHYGF (SEQ ID NO:9) 4 hours after vectoradministration in seven different organs. B: Distribution of genomesprovided by the wild-type AAV2 vector (upper panel), the control vectorAAV2-CVGSPCG (SEQ ID NO:21, middle panel) and AAV2-ESGHYGF (SEQ ID NO:9)lower panel). The control vector and wild-type vector accumulate in thereticuloendothelial system of the liver and spleen. AAV2-ESGHYGFaccumulates exclusively in the lungs. B: Comparison of the distributionof wild-type AAV2, control vector AAV2-CVGSPCG (SEQ ID NO:21) andAAV2-ESGHYGF (SEQ ID NO:9) in liver (upper panel), spleen (middle panel)and lung (lower panel). Mean values are shown with their standarddeviation. One-Way ANOVA. p<0.05=*; p<0.01=**; p<0.001=*** for n=3.

EXAMPLES

All data was determined as mean values±standard deviation (SD). Thestatistical analysis was performed using the GraphPad Prism 3.0 program(GraphPad Software, San Diego, USA). Data was analyzed by one-way ANOVAfollowed by multiple comparison tests as per Bonferroni. P values >0.05were considered significant.

Example 1: Selection of AAV2 Peptide Libraries

For the selection of tissue-specific AAV2 capsids, a random-displaypeptide library was prepared and selected in four rounds. A randomX₇-AAV peptide library with a theoretical diversity of 1×10⁸individually occurring clones was prepared using a two-stage protocol aspreviously described [26-27]. A degenerate oligonucleotide was firstproduced which codes for seven randomized amino acids at nucleotideposition 3967 in the AAV genome, which corresponds to the amino acidposition R588 in VP1. The oligonucleotide had the sequence:5′-CAGTCGGCCAGAGAGGC(NNK)₇GCCCAGGCGGCTGACGAG-3′ (SEQ ID NO: 11). Thesecond strand was produced using a Sequenase (Amersham, Freiburg,Germany) and the primer with the sequence 5′-CTCGTCAGCCGCCTGG-3′ (SEQ IDNO: 12). The double-stranded insert was cut with BglI, purified with theQIAquick Nucleotide Removal Kit (Qiagen, Hilden, Germany) and ligatedinto the library with SfiI digested library plasmid pMT187-0-3 [26]. Thediversity of the plasmid library was determined by the number of clonesgrown from a representative aliquot of transformed, electrocompetentDH5α bacteria on agar containing 150 mg/ml ampicillin. Library plasmidswere harvested and purified by using the Plasmid Preparation Kit fromQiagen. The AAV library genomes were packaged into chimeric wild-typeand library AAV capsids (AAV transfer shuttle) by transfecting 2×10⁸293T cells in 10 cell culture dishes (15 cm) with the plasmid pVP3 cm(containing the wild-type cap genes with modified codon usage withoutthe inverted terminal repeats) [27], the library plasmids and the pXX6helper plasmid [28], wherein the ratio between the plasmids was 1:1:2.The resulting AAV library transfer shuttles were used to infect 2×10⁸293T cells in cell culture dishes (15 cm) with an MOI of 0.5 replicationunits per cell. Cells were superinfected with Ad5 (provided by theLaboratoire de Therapie Génique, France), with an MOI of 5plaque-forming units (pfu/cell). The final AAV display library washarvested from the supernatants after 48 hours. The supernatants wereconcentrated using VivaSpin columns (Viva Science, Hannover, Germany)and purified by iodixanol density gradient ultracentrifugation aspreviously described [29], and titrated by real-time PCR using thecap-specific primers 5′-GCAGTATGGTTCTGTATCTACCAACC-3′ (SEQ ID NO: 13)and 5′-GCCTGGAAGAACGCCTTGTGTG-3′ (SEQ ID NO: 14) with the LightCyclersystem (Roche Diagnostics, Mannheim, Germany).

For the in vivo biopanning 1×10¹¹ particles of the genomic library wereinjected into the tail vein of FVB/N mice. The particles were given 8days for the distribution and the infection of the target cells. After 8days, the mice were killed and the lungs were removed. The total DNA ofthe tissue was extracted using the DNeasy Tissue Kit (Qiagen). Therandom oligonucleotides that were included in AAV particles of thelibrary and had accumulated in the tissue of interest were amplified bynested PCR using the primers 5′-ATGGCAAGCCACAAGGACGATG-3′ (SEQ ID NO:15) and 5′-CGTGGAGTACTGTGTGATGAAG-3′ (SEQ ID NO: 16) for the first PCRand the primers 5′-GGTTCTCATCTTTGGGAAGCAAG-3′ (SEQ ID NO: 17) and5-TGATGAGAATCTGTGGAGGAG-3′ (SEQ ID NO: 18) for the second PCR. ThePCR-amplified oligonucleotides were used to prepare secondary librariesfor three additional rounds of selection. The secondary libraries weregenerated like the primary libraries (see above), but without theadditional step of producing transfer shuttles. The secondary plasmidlibrary was used to transfect 2×10⁸ 293T cells in cell culture dishes(15 cm) at a ratio of 25 library plasmids per cell, wherein thetransfection reagent Polyfect (Qiagen) was used. After each round ofselection, several clones were sequenced. The applied selection methodis shown in FIG. 1.

Results:

After four rounds of selection, a total of 9 clones were sequenced. Thesequencing revealed that 5 clones had the peptide sequence ESGHGYF (SEQID NO: 2). Other clones showed the peptide sequences ADGVMWL (SEQ ID NO:3), GEVYVSF (SEQ ID NO: 4) and NNVRTSE (SEQ ID NO: 5). Three of the fourpeptide sequences, including the dominant clone ESGHGYF(SEQ ID NO: 2),as well as ADGVMWL(SEQ ID NO: 3) and GEVYVSF(SEQ ID NO: 4), displayed atleast one hydrophobic aromatic group. The peptides obtained in thevarious rounds of selection are shown in FIG. 2.

Example 2: Preparation and Quantification of Recombinant AAV Vectors

The clones enriched in Example 1 were produced as recombinant AAVvectors and tested for their transduction profile. Recombinant AAVvectors were produced by triple transfection of HEK293T cells. The cellswere incubated at 37° C., 5% CO₂ in Dulbecco's modified Eagle Medium(Invitrogen, Carlsbad, USA), supplemented with 1%penicillin/streptomycin and 10% fetal calf serum. Plasmid DNA wastransfected into 293T cells with the transfection agent Polyfect(Qiagen, Hilden, Germany). Four days after transfection, the cells wereharvested and lysed, and the vectors were purified by means of iodixanoldensity gradient ultracentrifugation as previously described [29]. Forthe transfections, pXX6 was used as adenoviral helper plasmid [28],which encodes the luciferase gene pUF2-CMV-luc [27] or the GFP genepTR-CMV-GFP [30], as was a plasmid encoding the AAV capsid of interest.The plasmids encoding the AAV capsid mutants which had been previouslyselected from the AAV library, and wild-type controls, were modifiedpXX2-187 [31] or pXX2 [28]. In addition, for an alanine scanning,further oligonucleotide inserts were made which encode modified variantsof the peptide ESGHGYF (SEQ ID NO: 2). The inserts were processed asdescribed into library inserts (see above). To quantify the recombinantvectors, the genomic titer was determined by the LightCycler system, aspreviously described [32], by real-time PCR using the CMV-specificprimers 5′-GGCGGAGTTGTTACGACAT-3′ (SEQ ID NO: 19) and5′-GGGACTTTCCCTACTTGGCA-3′ (SEQ ID NO: 20).

Example 3: Examination of the Tropism of the Recombinant AAV Vectors InVivo

To be able to examine the tropism of the enriched peptides in vivo, thepeptides were introduced into the capsid of a recombinant vectorcomprising a luciferase reporter gene. Vectors with mutated capsids wereinjected into mice along with control vectors. The AAV vectors wereadministered intravenously at a dose of 5×10¹⁰ vector genomes (vg)/mouse(n=3 animals per injected AAV clone). On day 14, the animals wereanesthetized with isoflurane. The luciferase expression was analyzedusing a Xenogen IVIS200 Imaging System (Caliper Lifescience, Hopkinton,USA) with the Living Image 4.0 (Caliper) software, followingintraperitoneal injection of 200 μl of luciferin substrate (150 mg/kg,Xenogen) per mouse. Representative, in vivo bioluminescence images ofthe expression of the transgene at different positions (ventral, dorsal,lateral) were taken when the luminescence in relative light units(photons/sec/cm2) reached the highest intensity. Then the animals weresacrificed, the organs of interest were removed quickly, and images ofthe expression of the transgene in individual organs were immediatelytaken. The organs were then frozen in liquid nitrogen and stored at −80°C. Three-dimensional reconstructions of the in vivo luminescence imageswere obtained by using the DLIT option of the Living Image 4 software,and the emitted light was measured in 5 different wavelengths from560-640 nm for three minutes each. To quantify the luciferaseexpression, the organs were homogenized in reporter lysis buffer (RLB,Promega, Madison, USA). The determination of the luciferase reportergene activity was carried out in a luminometer (Mithras LB9 40, BertholdTechnologies, Bad Wildbad, Germany) at 10-second intervals after theaddition of 100 μL luciferase assay reagent (LAR, Promega), with a2-second delay between each of the measurements. The values werenormalized in each sample with respect to the total amount of proteinusing the Roti NanoQuant protein assay (Roth, Karlsruhe, Germany).

Results:

It was found that the yield with respect to the vector titers forrecombinant vectors with luciferase reporter gene was comparable tovectors carrying a wild-type AAV2 capsid, which suggested that theenriched peptides do not adversely affect the assembly of the capsid orpackaging of the gene. The in vivo measurement of bioluminescence after14 days showed that the peptide ESGHGYF (SEQ ID NO: 2) led to a strongand lung-specific expression of the transgene (≤10⁵ p/sec/cm²/r). Theseresults were confirmed by the control experiments carried out ex vivowith explanted organs. A randomly selected control clone of thenon-selected library (CVGSPCG, SEQ ID NO:21) led to a weak geneexpression that occurred primarily in the heart and in some parts of theabdomen, but not in the lung. Wild-type AAV2 caused a weak geneexpression in the heart, liver and skeletal muscle, but not in thelungs. A three-dimensional reconstruction of bioluminescence imagesconfirmed the lung-specific expression. The peptide ADGVMWL (SEQ ID NO:3), which was also enriched during the in vivo selection, also led to alung-specific expression of the transgene, but was weaker than for thepeptide ESGHGYF(SEQ ID NO: 2). While the gene expression within 14 daysafter administration of the ADGVMWL (SEQ ID NO:3) luciferase vector wasvery low, it increased to about 5×10⁴ p/sec/cm²/r and could be observedspecifically in the lung 28 days after vector injection. These resultswere confirmed by the control experiments carried out ex vivo withexplanted organs. The investigation of the luciferase activity of tissuelysates from representative organs showed that wild-type AAV2 caused alow gene expression in the heart (2.9×10⁴ RLU/mg protein, see FIG. 3A,upper panel) and even lower levels of expression in other organs. Thecontrol peptide CVSGPCG (SEQ ID NO:21) produced a moderate geneexpression in the heart (8.7×10⁴ RLU/mg protein, see FIG. 3A, middlepanel). In contrast, vectors which had the lung specific ESGHGYF (SEQ IDNO:2) capsid led to a strong and specific gene expression in the lung(4.1×10⁵ RLU/mg protein, see FIG. 3A, lower panel). In the heart and inthe liver (i.e., in the two organs in which wild-type AAV2 and thepeptide vector CVGSPCG lead to a strong expression), the lung-specificESGHGYF (SEQ ID NO:2) vectors showed only an expression on the order ofthe background signal (about 1×10³ RLU/mg protein). In contrast, theexpression of the transgene in the lungs for the ESGHGYF (SEQ ID NO:2)vectors was more than 200-fold higher than the expression mediated bywild-type AAV2 or by the CVGSPCG (SEQ ID NO:21) control vectors (seeFIG. 3B).

The results further showed that the lung-specific expression of thetransgene mediated by the ESGHGYF (SEQ ID NO:2) vectors remainedorgan-specific over a long period. After intravenous administration ofthe lung-specific AAV2 ESGHGYF (SEQ ID NO:2) luciferase vectors, theexpression of the transgene was measured over a period of 164 days. Theradiation emitted in the lung region was determined quantitatively. Overthe entire period of time, the expression of the transgene was stable ata high level, and was limited to the lung. The lowest expression in thelung was measured at day 7, a peak was reached on day 42, and theradiation declined only slowly to the last measurement on day 164 (FIG.4).

Example 4: Alanine Scanning for the Peptide ESGHGYF (SEQ ID NO: 2)

To investigate the importance of the individual amino acids in thepeptide ESGHGYF (SEQ ID NO:2) in relation to the lung specificity, analanine scanning was performed.

Results:

It was found that the lung-specific tropism was not changed by replacingthe first two amino acids. However, if amino acids 3-4 or 5-7 wereexchanged, there was either a total loss of infectivity (position 3 or4) or a change in specificity to heart or skeletal muscle (positions5-7).

Example 5: Analysis of the Vector Distribution

In order to check whether the lung-specific expression of the transgeneof intravenously injected ESGHGYF (SEQ ID NO:2) vectors is based on alung-specific homing, first the distribution of vectors was investigatedfour hours after intravenous administration of 5×10¹⁰ gp/mouse. Thequantification of the vector genomes was performed by real-time PCR.First, the total DNA was extracted from the organ concerned at varioustime points after intravenous administration of 5×10¹⁰ vg/mouse using atissue homogenizer (Precellys 24, Peqlab, Erlangen, Germany) and theDNeasy Tissue Kit (Qiagen, Hilden, Germany) according to themanufacturer's instructions. The DNA was quantified using aspectrophotometer (NanoDrop ND-2000C, Peqlab). The analysis of the AAVvector DNA in the tissues was performed by quantitative real-time PCRusing the above-described CMV-specific primer, wherein 40 ng of templatewere used, normalized with respect to the total DNA.

Results:

The quantification of the vector genomes by real-time PCR showed alung-specific homing of ESGHGYF (SEQ ID NO:2). The amount of vectorgenomes which could be detected in the lungs (3.8×10⁵±1.9×10⁵ vg/100 ngtotal DNA) was about 6-100 times higher than the amount of vectorgenomes which was demonstrated in another organ (FIG. 5A). To determinethe direct correlation between vector homing and expression of thetransgene, the vector distribution of wild-type AAV2, the controlpeptide CVGSPCG and the lung-specific peptide ESGHGYF (SEQ ID NO:2) wasmeasured 14 days after intravenous administration of 5×10¹⁰ gp/mouse,i.e., at the time when the expression of the transgene was determined(see above). The genomes provided by wild-type AAV2 vectors were mainlyrecovered from the liver and spleen, and the genomes of vectors whichhad the control peptide were obtained largely from the spleen. In total,the amount of vector genomes which were detected in the spleen wererelatively equal (4×10³ vp/100 ng of total DNA) in all examined capsidvariants, suggesting a nonspecific capture mechanism for the particlesin the reticuloendothelial system which is independent of the provisionand the expression of the transgene. In contrast, the distribution dataof genomes which were provided by vectors which had the lung-specificpeptide ESGHGYF (SEQ ID NO:2) was highly similar to the expression dataof the transgene, with a highly specific accumulation observed in thelungs. The amount of vectors detected in the lung which showed thepeptide ESGHGYF (SEQ ID NO: 2) was about 250-fold higher than in otherorgans, and up to 500-fold higher than in lungs which were injected witha wild-type vector or a control capsid vector (FIG. 5B). The samedistribution values between the organs were found 28 days after vectoradministration. The direct comparison between the three vector capsidvariants for the quantities of genomes found is shown in FIG. 5C for thethree tissues in which relevant amounts of vector DNA were detected.Overall, this data indicates that a lung-specific expression of thetransgene, mediated by ESGHGYF (SEQ ID NO: 2) vectors, is achieved by atissue-specific homing of circulating particles.

Example 6: Immunohistochemistry and Histology

Immunohistochemistry was used to visualize the expression of thetransgene at the cellular level in the lung, as well as in a controlorgan, 14 days after the intravenous administration of the rAAV-GFPvector having the peptide ESGHGYF (SEQ ID NO:2) and/or the wild-type AAVcapsid as control. The lungs of the animals were fixed ex situ with 4%(w/v) paraformaldehyde via the trachea under hydrostatic pressure of 20cm of water for 20 minutes, followed by 24 hours of immersion in thesame fixative. The lung tissues were embedded in paraffin. Sections witha thickness of 2 μm were removed from wax, rehydrated and used forimmunohistochemistry. An immunohistochemical procedure was performedusing polyclonal antibodies for GFP (A-11122, Invitrogen) or CD31(AB28364, Abcam, Cambridge, USA). Endogenous peroxidase was inactivatedwith 1% H₂O₂ in methanol for 30 minutes. Prior to staining with CD31,the sections were heated in citrate buffer (pH 6.0) for 20 minutes at100° C. After washing in PBS, the sections were incubated for 30 minuteswith PBS, 10% goat serum (Vector Lab, Burlingame, USA) and 2% milkpowder (Roth). Primary antibodies were allowed to bind for 1 hour at 37°C. After washing in PBS, the sections were incubated for 30 minutes witha secondary, biotinylated goat anti-rabbit antibody (Vector Lab). Boundantibodies were visualized by using the VECTASTAIN-Elite ABC kit (VectorLab) and 3,3′-diaminobenzidene (DAB, Sigma-Aldrich, St. Louis, USA).Selected sections were counterstained with Hemalum.

Results:

In the lungs of mice injected with rAAV-ESGHGYF (SEQ ID NO:9), amicroscopic examination showed intensive staining of the endothelialcells over the entire pulmonary micro-vasculature and to a slightlylesser extent in the large pulmonary vessels (data not shown). Incontrast, pulmonary tissue of mice which was injected with wild-typeAAV2 vector showed no staining. To confirm the tissue specificity, theliver was analyzed as a control organ (a tissue which is known tofrequently demonstrate high expression of a transgene) after injectionof wild-type AAV2 vector. In the liver, hepatocyte staining was observedafter administration of wild-type rAAV2 vector; but no staining wasobserved after administration of rAAV2-ESGHGYF (SEQ ID NO:9) vector. Theendothelial lineage of pulmonary cells transduced with the vectors wasconfirmed by CD31 staining, wherein the pattern obtained by the GFPstaining was confirmed in serial sections of the lungs of mice injectedwith rAAV2-ESGHGYF (SEQ ID NO:9) (data not shown).

READINGS

-   [1] Barst et al., 2004 J Am Coll Cardiol, 43: 40-47-   [2] McLaughlin et al., 2009, Circulation, 119: 2250-2294-   [3] Stenmark et al., 2009, Am J Physiol Lung Cell Mol Physiol, 297:    1013-1032-   [4] Friedman et al., 2012 J Obes, 2012: 505274-   [5] Chin et al., 2005 Coron Artery Dis., 16: 13-18.-   [6] Hemnes et al., 2008 Int J Clin Pract Suppl: 11-19-   [7] Humbert et al., 2009 Am J Respir Crit Care Med, 179: 650-656-   [8] Simonneau et al., 2009 J Am Coll Cardiol, 54: 43-54-   [9] Humbert et al., 2006, Am J Respir Crit Care Med, 173: 1023-1030-   [10] Tenenbaum et al. 2003, Curr Gene Ther 3: 545-565-   [11] Work et al., 2006, Mol Ther, 4: 683-693-   [12] Shi et al., 2006 Hum Gene Ther, 17: 353-361-   [13] US 2007/0172460 A1-   [14] Michelfelder et al., 2009 PLOS one, 4(4): e5122-   [15] Shi and Bartlett, 2003, Mol Ther, 7: 515-525-   [16] Loiler et al., 2003, Gene Ther, 10: 1551-1558-   [17] Rabinowitz et al., 1999, Virology, 265: 274-285-   [18] Wu et al., 2000, J Virol, 74: 8635-8647-   [19] Shi et al., 2001, Hum Gene Ther, 12: 1697-1711-   [20] Warrington et al. 2004, J Virol, 78: 6595-6609-   [21] Girod et al. 1999, Nat Med, 5: 1052-1056-   [22] Grifman et al. 2001, Mol Ther, 3: 964-975-   [23] Opie et al., 2003, J Virol, 77: 6995-7006-   [24] Kern et al., 2003, J Virol, 77: 11072-11081-   [25] Russell et al., 1998, J Virol, 72: 309-319-   [26] Muller et al., 2003, Nat Biotechnol 21, 1040-1046-   [27] Water Kamp, et al., 2006 J Gene Med 8, 1307-1319-   [28] Xiao et al., 1998, Journal of Virology 72, 2224-2232-   [29] Zolotukhin, et al., 1999, Gene Ther 6, 973-985-   [30] McCarty et al., 2001, Gene Ther 8, 1248-1254-   [31] Michelfelder, et al., 2007, Exp Hematol 35, 1766-1776-   [32] Rohr et al., 2005 J Virol Methods 127, 40-45

The invention claimed is:
 1. A capsid protein of a viral vector comprising the amino acid sequence of SEQ ID NO:
 1. 2. The capsid protein according to claim 1, which has the amino acid sequence of SEQ ID NO: 2, or a variant thereof which differs from the amino acid sequence of SEQ ID NO: 2 by a modification of one or both of the amino acids located in positions 1 and 2 of the N-terminus of the amino acid sequence of SEQ ID NO:2.
 3. The capsid protein according to claim 1, which is a capsid protein of an adeno-associated virus (AAV).
 4. The capsid protein according to claim 3, which is a capsid protein of an AAV of a serotype selected from the group consisting of serotypes 2, 4, 6, 8, and
 9. 5. The capsid protein according to claim 4, which is a capsid protein of an AAV of serotype
 2. 6. The capsid protein according to claim 5, which is a VP1 protein of an AAV of serotype
 2. 7. The capsid protein according to claim 1, wherein the amino acid sequence of SEQ ID NO:1 is present in the region of amino acids 550-600 of the capsid protein.
 8. The capsid protein according to claim 1, comprising the amino acid sequence of SEQ ID NO:
 9. 9. A nucleic acid which encodes a capsid protein according to claim
 1. 10. A plasmid which comprises a nucleic acid according to claim
 9. 11. A recombinant viral vector which comprises a capsid and a transgene packaged therein, wherein the capsid comprises at least one capsid protein comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof which differs from the amino acid sequence of SEQ ID NO: 2 by modification of one or both of the amino acids located in positions 1 and 2 of the N-terminus of the amino acid sequence of SEQ ID NO:
 2. 12. The recombinant viral vector according to claim 11, which is a recombinant AAV vector.
 13. The recombinant AAV vector according to claim 12, which is an AAV vector of a serotype selected from the group consisting of serotypes 2, 4, 6, 8, and
 9. 14. The recombinant AAV vector according to claim 13, which is an AAV vector of serotype
 2. 15. The recombinant AAV vector according to claim 11, wherein the transgene encodes a nitric oxide synthase or the bone morphogenic protein receptor 2 (BMPR2).
 16. The recombinant AAV vector according to claim 15, wherein the transgene encodes endothelial nitric oxide synthase (eNOS) or inducible nitric oxide synthase (iNOS).
 17. The recombinant AAV vector according to claim 11, wherein the transgene is in the form of an ssDNA or a dsDNA.
 18. The recombinant AAV vector according to claim 11, for use in a method for the treatment of a lung disorder or a lung disease in a subject.
 19. The recombinant AAV vector for use in a method according to claim 18, wherein the lung disease is pulmonary hypertension or pulmonary arterial hypertension.
 20. The recombinant AAV vector for use in a method according to claim 18, wherein the subject is a mammal.
 21. The recombinant AAV vector for use in a method according to claim 18, wherein the vector is formulated for intravenous administration.
 22. A cell which comprises a capsid protein according to claim
 1. 23. A pharmaceutical composition which comprises a capsid protein according to claim
 1. 24. The nucleic acid of claim 9, wherein the capsid protein comprises the amino acid sequence of SEQ ID NO:9.
 25. The recombinant viral vector of claim 11, wherein the capsid protein comprises the amino acid sequence of SEQ ID NO:9. 